的使用<em>前体内</em>钱德勒循环装置，以评估改性聚合物血导管的生物相容性
Summary
Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.
Abstract
当合成的表面被引入到主体时的异物反应。它的特征在于，吸附血液中的蛋白质，并随后附着的血小板活化，单核细胞/巨噬细胞的粘附性，和炎性细胞信号转导事件，导致后并发症。钱德勒循环设备是一个实验系统，让研究人员研究时，大量血液灌流与聚合物管道发生的分子和细胞的相互作用。为此，该装置已被用作离体模型允许的各种聚合物表面修饰的抗炎性质的评估。我们实验室已经表明，血液导管，通过光敏化学与重组CD47的共价修饰，可以赋予生物相容性的聚合物表面。附加CD47到聚合物表面可以促进聚合血液导管的疗效的有效手段。她EIN是该方法详述用于追加重组CD47对临床相关的聚合血液的导管和使用钱德勒回路作为体外实验模型研究与CD47的改性和控制导管血液相互作用的光活化的化学反应。
Introduction
许多临床程序，如体外循环，肾透析，需要使用聚合血导管，并经常与后并发症相关联。当与血液灌流，这些聚合物非法的异物反应（FBR），从而导致吸附血液中的蛋白和血小板，单核细胞/巨噬细胞的粘附，以及促炎细胞因子，所有这些都有助于后并发症和释放/或设备故障2,3。因此，策略来解决这一问题保持生物材料研究的重要和持续的区域。研究者已经试图通过改变血液接触的表面具有生物活性或生物惰性分子4-6来解决这个问题。研究在我们的实验室都集中在所附的重组CD47（recCD47）到聚合物的生物材料作为一种策略，以减轻FBR和提高这些材料的功效。 CD47是一种广泛表达transmembrANE蛋白与免疫逃逸时表达细胞7-10显示了一个著名的角色，授予“自我”的状态，在承诺时追加到聚合物表面11-13授予的生物相容性。信号调节蛋白α（SIRPα），同族受体CD47和免疫受体基于酪氨酸的抑制基序（ITIM）基家族的跨膜蛋白的成员，表达于骨髓来源14的细胞。我们以前表明，CD47，通过SIRPα介导的细胞信号传导，下调在体外 ， 离体 ，以聚氨酯（PU）和聚氯乙烯（PVC）的免疫反应，并且在体内模型11-13。
中央到我们的调查是一种相对较新的光活化化学，本文中所描述，其中的化学反应性硫醇基团通过管路与多官能聚合物（PDT-BZ反应共价地附于聚合管PH），2 -吡啶基二硫（PDT）组成，所述光反应性二苯甲酮（BzPh）和羧基改性聚烯丙基胺11-13。降低了共价附PDT组与三（2 -羧乙基）膦盐酸盐（TCEP）11产生一个硫醇化表面，其可接着与治疗性结构部分的反应。这里和前面详述12,13，recCD47，进一步修饰与另外的C末端多聚赖氨酸尾12,13，与磺基琥珀酰亚胺基-4- [N -maleimidomethyl]环己烷-1 -羧酸乙酯反应（磺基-SMCC） 1小时，以产生硫醇反应性基团，从而允许管道和recCD47 11之间的硫化物键的形成。的CD47官能表面的抗炎能力进行了测试， 当然VIV O，使用钱德勒回路装置与人全血，它最初是于1958年描述为血栓凝固15的体外模型。该装置依靠闭管系统部分地填充有空气和旋转马达来循环血液通过导管15。此实验模型提供了机会，检查血液接触后修饰和未修饰的表面的效果，以及这些表面修饰后，血液中的细胞的生理学效果。
recCD47可以被附加到各种使用这种光活化化学聚合的表面，并且它的抗炎能力可以通过利用临床相关的体外模型模仿血液灌注过的聚合物表面11,12进行评估。改性recCD47临床级血液导管显示显著少血小板和炎性细胞附着比未改性的聚合物时，在该装置暴露于人血。这个修改过程中的一步一步的说明详述如下。
Protocol
Representative Results
Discussion
光活化化学（总结于图1），使得几乎任何聚合物表面具有足够的烃，以方便PDT-BzPh附着和随后的UV照射，以修改光激活的PDT-BzPh。官能聚合物与表面活性巯基可以为后续的连接各种兴趣测试的分子组成。在我们特定的研究中，我们选择了重组CD47 11-13。 ，我们使用所涉及的蛋白质的胺基团与双官能交联剂SMCC 11-13的反应的特定结合化学。这提供硫醇反应recCD47用于与巯基?…
Divulgazioni
The authors have nothing to disclose.
Materials
| Name | Company | Catalog Number | Comments |
| 16% Paraformaldehyde (PFA) | Thermo Scientific | 58906 | Caution! Use in fume hood |
| 25% Glutaraldehyde | VWR | AAA17876-AP | Caution! Use in fume hood |
| 2-pyridyldithio,benzophenone (PDT-BzPH) | Synthesized in lab | N/A | |
| Bovine Serum Albumin (BSA) | Sigma | A3059-100G | |
| Citrate | Sigma | S5770-50ML | |
| Digital Camera | Leica | DC500 | Out of production |
| Dimethylformamide (DMF) | Sigma | 270547-100ML | Caution! Use in fume hood |
| Dulbecco’s Phosphate Buffered Saline (DPBS) | Gibco/Life Technologies | 14190-136 | |
| Fluorescent Microscope | Nikon | TE300 | |
| Glacial Acetic Acid | Fisher Scientific | A38-212 | Caution! Use in fume hood |
| Human CD47 (B6H12) – FITC Antibody | Santa Cruz Biotechnology | SC-12730 | |
| Osmium Tetroxide | Acros Organics | 197450050 | Caution! Use in fume hood |
| Potassium Bicarbonate (KHCO3) | Sigma | 237205-100G | |
| Potassium Phosphate Monobasic (KH2PO4) | Sigma | P5655-100G | |
| PVC Tubing (Cardiovascular Procedure Kit) | Terumo Cardiovascular Systems | 60050 | Most clinical-grade tubing will work |
| Scanning Electron Microscope | JEOL | JSM-T330A | |
| Sodium Chloride (NaCl) | Fisher Scientific | BP358-212 | |
| Microplate Reader | Molecular Devices | Spectramax Gemini EM | |
| Sulfo-SMCC | Sigma | M6035-10MG | Moisture Sensitive! |
| tris (2-carboxyethyl) phosphine (TCEP-HCl) | Thermo Scientific | 20491 | |
| Tris Base | Sigma | T1503-100G | |
| Tween-20 | Bio-Rad | 170-6531 | |
| Vectashield with DAPI | Fisher Scientific | H-1200 | Light sensitive! |
| Zeba Spin Desalt Columns – 7K MWCO | Thermo Scientific | 89891 |
Riferimenti
- Bruck, S. D. Medical applications of polymeric materials. Med. Prog. Technol. 9 (1), 1-16 (1982).
- Anderson, J. M., Rodriguez, A., Chang, D. T. Foreign body reaction to biomaterials. Semin. Immunol. 20 (2), 86-100 (2008).
- Levy, J. H., Tanaka, K. A. Inflammatory response to cardiopulmonary bypass. Ann. Thorac. Surg. 75, S715-S720 (2003).
- Sperling, C., Maitz, M. F., Talkenberger, S., Gouzy, M. F., Groth, T., Werner, C. In vitro blood reactivity to hydroxylated and non-hydroxylated polymer surfaces. Biomaterials. 28, 3617-3625 (2007).
- Sperling, C., Schweiss, R. B., Streller, U., Werner, C. In vitro hemocompatibility of self-assembled monolayers displaying various functional groups. Biomaterials. 26, 6547-6457 (2005).
- Vasita, R., Shanmugam, I. K., Katt, D. S. Improved biomaterials for tissue engineering applications: surface modification of polymers. Curr. Top. Med. Chem. 8, 341-353 (2008).
- Subramanian, S., Parthasarathy, R., Sen, S., Boder, E. T., Discher, D. E. Species- and cell type-specific interactions between CD47 and human SIRPalpha. Blood. 107 (6), 2548-2556 (2006).
- Tsai, R. K., Discher, D. E. Inhibition of ‘self’ engulfment through deactivation of myosin-II at the phagocytic synapse between human cells. J Cell Biol. 180 (5), 989-1003 (2008).
- Berg, T. K., vander Schoot, C. E. Innate immune ‘self’ recognition: a role for CD47-SIRPalpha interactions in hematopoietic stem cell transplantation. Trends Immunol. 29 (5), 203-206 (2008).
- Oldenborg, P. A., Zheleznyak, A., Fang, Y. F., Lagenaur, C. F., Gresham, H. D., Lindberg, F. P. Role of CD47 as a marker of self on red blood cells. Science. 288 (5473), 2051-2054 (2000).
- Stachelek, S. J., et al. The effect of CD47 modified polymer surfaces on inflammatory cell attachment and activation. Biomaterials. 32 (19), 4317-4326 (2001).
- Finley, M. J., Rauva, L., Alferiev, I. S., Weisel, J. W., Levy, R. J., Stachelek, S. J. Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces. Biomaterials. 33, 5803-5811 (2012).
- Finley, M. J., Clark, K. A., Alferiev, I. S., Levy, R. J., Stachelek, S. J. Intracellular signaling mechanisms associated with CD47 modified surfaces. Biomaterials. 34, 8640-8649 (2013).
- Ravetch, J. V., Lanier, L. L. Immune inhibitory receptors. Science. 290, 84-89 (2000).
- Chandler, A. B. In vitro thrombotic coagulation of blood: a method for producing a thrombus. Lab Invest. 7, 110-114 (1958).
- Thorsen, T., Klausen, H., Lie, R. T., Holmsen, H. Bubble-induced aggregation of platelets: effects of gas species, proteins, and decompression. Undersea Hyperb Med. 20 (2), 101-119 (1993).
- Ritz-Timme, S., Eckelt, N., Schmidtke, E., Thomsen, H. Genesis and diagnostic value of leukocyte and platlet accumulations around “air bubbles” in blood after venous air embolism. Intl J of Legal Med. 111 (1), 22-26 (1998).
- Miller, R., Fainerman, V. B., Wüstneck, R., Krägel, J., Trukhin, D. V. Characterization of the initial period of protein adsorption by dynamic surface tension measurements using different drop techniques. Colloids and Surfaces A. 131 (1-3), 225-230 (1998).
- Oeveren, W. V., Tielliu, I. F., de Hart, J. Comparison of modified chandler, roller pump, and ball valve circulation models for in vitro testing in high blood flow conditions: application in thrombogenicity testing of different materials for vascular applications. Int J Biomater. 2012, (2012).
